Therapeutic vaccine for the treatment of papillomavirus lesions

ABSTRACT

An attenuated vaccinia virus GAB-1 and its use in a vaccine for treatment of papillomavirus lesions is described. In preferred embodiments, the Lederle-Chorioallantoic strain of vaccinia virus is serially passaged in chicken embryo-fibroblast (CEF) cells by at least 100 passages. GAB-1 has reduced virulence and is safe to use without side effects after attenuation by serial passaging, but remains highly immunogenic. Experimentation has found that GAB-1 is much more immunogenic than other strains of vaccinia virus, including Western Reserve (WR) and modified Vaccinia Ankara (MVA). GAB-1 can be used safely in humans for treating tumorous lesions caused by human papillomavirus (HPV).

This application is a continuation of U.S. patent application Ser. No.16/598,516 filed on Oct. 10, 2019. These and all other referencedextrinsic materials are incorporated herein by reference in theirentirety. Where a definition or use of a term in a reference that isincorporated by reference is inconsistent or contrary to the definitionof that term provided herein, the definition of that term providedherein is deemed to be controlling.

FIELD OF THE INVENTION

The field of the invention is attenuated viral vaccine.

BACKGROUND

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Human papillomavirus (HPV) is a group of viruses that spread throughsexual contact. Some types of HPVs (e.g., HPV-6 and -11) cause genitalwarts that rarely turn into cancer, while other types (e.g., HPV-16,-18, -31, and -45) can cause cervical, oral, and anal cancer. Warts andpre-cancerous lesions caused by HPV can be treated by cryosurgery,laser, loop electrosurgical excision procedure (LEEP), or surgery.However, these treatments are invasive, and do not cure the viralinfection. Although vaccines are available for certain types of HPV,vaccines do not cure cancer or warts caused by HPV.

U.S. Pat. No. 6,582,693 to the inventor in the current applicationteaches a recombinant vaccinia virus derived from the modified vacciniaAnkara (MVA) encoding and capable of expressing the E2 gene of Bovinepapillomavirus, and the use of the virus in the treatment of lesionscaused by papillomavirus. However, the attenuated MVA virus has lowyields of virus production. U.S. Pat. No. 10,105,436 to Szalay et al.teaches the use of about a dozen strains of vaccinia virus (e.g., NewYork City Board of Health, Dryvax, ACAM1000, ACAM2000, Lister, EM63,LIVP, Tian Tan, Copenhagen, Western Reserve, and MVA) to induce ananti-tumor immune response. However, Szalay does not teach a strain ofvaccinia virus that is more immunogenic.

Thus, there is still a need for a new attenuated vaccinia virus vectorthat is safe and more immunogenic.

All publications identified herein are incorporated by reference to thesame extent as if each individual publication or patent application werespecifically and individually indicated to be incorporated by reference.Where a definition or use of a term in an incorporated reference isinconsistent or contrary to the definition of that term provided herein,the definition of that term provided herein applies and the definitionof that term in the reference does not apply.

SUMMARY OF THE INVENTION

The inventive subject matter provides compositions and methods in whicha new, highly attenuated vaccine is derived from theLederle-Chorioallantoic strain of vaccinia virus by serial passaging.Unexpectedly, the attenuated Lederle-Chorioallantoic strain, while beingsafe to use (i.e., less virulent), is highly immunogenic. It iscontemplated that it can be used in the treatment of infectious diseases(e.g., small pox) and tumorous lesions (e.g., papillomavirus (HPV)).

In some embodiments, the attenuated Lederle-Chorioallantoic strainsproduce a higher antibody titer than wildtype Western Reserve strain ofvaccinia virus in an animal. The wildtype Western Reserve strain ofvaccinia virus is the most virulent strain of vaccinia virus in animalmodels. A widely used wild type Western Reserve strain of vaccinia virusis ATCC VR1354; NCBI Ref. Seq: NC-006998.1. It is neurotropic in miceand more virulent than the Wyeth strain. It is lethal to an animalincluding human, if 10⁷ or more viral particles are given. In preferredembodiments, the attenuated Lederle-Chorioallantoic strains can generateat least 100 times as many viral particles as the MVA strain. Inespecially preferred embodiments, the attenuated Lederle-Chorioallantoicstrains can generate 1000 times as many viral particles compared withthe MVA strain.

In some embodiments, the attenuated Lederle-Chorioallantoic strainsproduce a higher antibody titer than a Modified Vaccinia virus Ankara(MVA) E2 strain of vaccinia virus in a patient. The MVA E2 strain wasfound to be highly effective against HPV induced lesions. See U.S. Pat.No. 6,582,693 to Rosales. In preferred embodiments, attenuatedLederle-Chorioallantoic strains can generate at least twice as manyantibodies as the MVA strain. In especially preferred embodiments, theattenuated Lederle-Chorioallantoic strains can generate four times viralparticles compared with the MVA strain.

The attenuated Lederle-Chorioallantoic strains can stimulate the immunesystem by inducing the host to generate antibodies and cytotoxic cellscapable of eliminating tumour lesions caused by viral infections. Thetumour cells are destroyed by apoptosis and/or are recognized by immunecells after local injection. It is contemplated that the attenuatedLederle-Chorioallantoic strains can be used to treat lesions caused bypapillomavirus located in the throat, cervix, bladder, lungs, orprostate in individuals infected with oncogenic and non-oncogenicpapillomaviruses, as well as different types of tumours that are locatedvisually and internally located by tomography and X-rays.

In preferred embodiments, the attenuated Lederle-Chorioallantoic strainis created by attenuating the original Lederle strain of vaccine virusby serial passages in chicken embryo fibroblasts cells (CEF). It iscontemplated that other host cells can be used to serially passage theattenuated Lederle-Chorioallantoic strain, for example, a monkey kidneycell line (VERO), and a monkey epithelial cell line (BSC-1). It furthercontemplated multiple host cells can be used in combination to seriallypassage the attenuated Lederle strain of vaccine virus.

After serial passaging, the Lederle virus becomes highly attenuated. TheLederle virus strain can be attenuated by cultured into the CEF cellsand passaged over multiple times, for example, 50, 100, 200 times, ormore, in order to obtain a highly attenuated and highly immunogenicvirus. In some embodiments, the attenuated Lederle strain loses at least5,000 nucleotides from its genome during serial passaging. In preferredembodiments, the attenuated Lederle strain loses between 8,000 and12,000 nucleotides from its genome during serial passaging.

GAB-1 is an attenuated Lederle-Chorioallantoic strain of vaccinia virusafter being passaged 200 times in CEF. The safety of the GAB-1 virus hasbeen tested in animal models in which no adverse effects were observed.The GAB-1 virus can be used for treatment of HPV-associated lesions,including warts and precancerous and cancerous lesions found on thecervix, penis, anus, and throat, as well as use in other types ofcancers (for example, bladder, prostate, melanoma, and lungs). The GAB-1virus can be used for the treatment of both superficial and internallesions and can be applied locally. It is shown that the GAB-1 virus canlead to the remission of carcinogenic lesions caused by papillomavirus.In addition, the GAB-1 virus can be used to cause regression of HPVlesions and different types of tumours without the possible presence ofintegrated viruses.

Experiments in human patients have shown that local injection of GAB-1causes regression of pre-cancerous lesions. Patients presentingpapillomavirus lesions showed a reduction up to complete elimination oflesions after treatment with the GAB-1 virus. These results demonstratethat GAB-1 virus is capable of arresting tumour growth. Moreover,animals immunized with GAB-1 are protected against infection by wildtype Western Reserve (WR) vaccinia virus (i.e., no signs of sickness areobserved). It is further contemplated that the GAB-1 virus can protectanimals against poxvirus infections, for example, smallpox.

The present inventive subject matter also provides a pharmaceuticalcomposition or formulation of the GAB-1 virus in a pharmaceuticallyacceptable carrier or diluent. In some embodiments, the GAB-1 virus isused to prepare an injectable solution for use in humans. It iscontemplated that the pharmaceutical composition can be used toeliminate any type of pre-cancer and cancer lesions generated bypapillomavirus. In some embodiments, the pharmaceutical composition canbe inoculated directly into tumours or lesions, including superficiallesions, and internal lesions observable to the naked eye or through atomography study or X-ray analysis. It is contemplated that thecomposition is used in the treatment of all types of human tumours. Inpreferred embodiments, the composition is used to cure lesions caused bypapillomavirus infections.

In some embodiments, the pharmaceutical composition has an adjuvant thathelps create a stronger immune response in the subject. Contemplatedadjuvants include amorphous aluminum hydroxyphosphate sulfate (AAHS),aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate(Alum), AS04 (Monophosphoryl lipid A (MPL)+aluminum salt), MF59 (Oil inwater emulsion composed of squalene), AS01B (Monophosphoryl lipid A(MPL) and QS-21, a natural compound extracted from the Chilean soapbarktree, combined in a liposomal formulation), CpG 1018 (Cytosinephosphoguanine (CpG), a synthetic form of DNA that mimics bacterial andviral genetic material). In preferred embodiments, the adjuvant is analuminum salt, for example, aluminum hydroxide, aluminum phosphate, andaluminum potassium sulfate, or any combination thereof.

The inventive subject matter also provides a method of immunization,comprising administering a pharmaceutical composition to a subject. Inpreferred embodiments, the pharmaceutical composition comprises aLederle-Chorioallantoic strain of vaccinia virus has been seriallypassaged in chicken embryo fibroblast cells (CEF) by at least 200passages. In some embodiments, administering the pharmaceuticalcomposition comprising injecting the pharmaceutical composition havingbetween 10⁴ and 10⁶ viral particles into a subject. In preferredembodiments, no adjuvant is added because vaccinia virus does not needadditional adjuvants, as it tends to act as an adjuvant by itself,making it a good vector to make vaccines.

In some embodiments, the subject is at least partially protected frominfection by smallpox virus after administration of the pharmaceuticalcomposition. In preferred embodiments, the subject is fully protectedfrom infection by smallpox virus after administration of thepharmaceutical composition. In some embodiments, the subject is at leastpartially protected from infection by Reserve virus (WR) afteradministration of the pharmaceutical composition. In preferredembodiments, the subject is fully protected from infection by Reservevirus (WR) after administration of the pharmaceutical composition. Inpreferred embodiments, the subject does not develop adverse side effectsafter administration of the pharmaceutical composition. In preferredembodiments, the subject is a human.

The inventive subject matter provides a method of treating cancer,comprising administering a pharmaceutical composition to a subject. Inpreferred embodiments, the pharmaceutical composition comprises aLederle-Chorioallantoic strain of vaccinia virus has been seriallypassaged in chicken embryo fibroblast cells (CEF) by at least 200passages. In some embodiments, administering the pharmaceuticalcomposition comprising injecting the pharmaceutical composition havingbetween 10⁴ and 10⁶ viral particles into a lesion in the subject. Inpreferred embodiments, the lesion is caused by human papillomavirus(HPV). To be considered effective, the lesion has at least 80% reductionis size, up to 100% eradication 14 weeks after treatment.

It is contemplated that the lesion is in a cervix, anus, penis, orlarynx of the subject. It is contemplated that the pharmaceuticalcomposition stimulates the immune system of an animal organism togenerate antibodies directed towards cancerous cells. It is alsocontemplated that the pharmaceutical composition stimulates the immunesystem of an animal organism to activate cytotoxic cells to kill tumourcells. The composition can be used in animal organisms, specifically inhumans.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing figures in which like numerals represent like components. whichare responsible for the regression and elimination of precancerous andcancerous lesions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C depict aspects of GAB-1 virus infection in cellculture, compared with wildtype Western Reserve (WR) virus. FIG. 1A is achart of viral yield of GAB-1 and WR viruses in chicken embryofibroblasts cells (CEF), a monkey kidney cell line (VERO) and a monkeyepithelial cell Line BSC-1. FIG. 1B shows viral plaques of WR and GAB-1viruses in VERO and BSC-1 cells. FIG. 1C shows viral plaques of WR orGAB-1 viruses in CEF.

FIG. 2 shows the DNA molecular weight of different isolates from Lederlevirus passages over the CEF cells.

FIG. 3A shows a pustule developed in the upper arm in a monkey 7 daysafter injection with GAB-1 virus, but not in a monkey injected with PBS.FIG. 3B shows GAB-1 vaccine fully protected monkeys against challengewith the WR virus.

FIG. 4 is a colposcopy showing the disappearance of intraepitheliallesion in patients treated with GAB-1.

FIG. 5 is chart showing the viral yield of GAB-1 virus compared with MVAE2 recombinant vaccine in CEF cells.

Table I is a chart comparing monkeys immunized with GAB-1 and a controlmonkey injected with PBS buffer.

Table II is a chart further analyzing the antibody levels in monkeys inTable I.

Table III is a chart comparing antibody levels in patients immunizedwith GAB-1 or MVA E2.

DETAILED DESCRIPTION

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise.

Unless the context dictates the contrary, all ranges set forth hereinshould be interpreted as being inclusive of their endpoints, andopen-ended ranges should be interpreted to include only commerciallypractical values. Similarly, all lists of values should be considered asinclusive of intermediate values unless the context indicates thecontrary.

The recitation of ranges of values herein is merely intended to serve asa shorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value with a range is incorporated into the specification asif it were individually recited herein. All methods described herein canbe performed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

The following discussion provides many example embodiments of theinventive subject matter. Although each embodiment represents a singlecombination of inventive elements, the inventive subject matter isconsidered to include all possible combinations of the disclosedelements. Thus, if one embodiment comprises elements A, B, and C, and asecond embodiment comprises elements B and D, then the inventive subjectmatter is also considered to include other remaining combinations of A,B, C, or D, even if not explicitly disclosed.

As used herein, and unless the context dictates otherwise, the term“coupled to” is intended to include both direct coupling (in which twoelements that are coupled to each other contact each other) and indirectcoupling (in which at least one additional element is located betweenthe two elements). Therefore, the terms “coupled to” and “coupled with”are used synonymously.

In Table I, monkeys 1 and 3-5 are vaccinated with GAB-1, while controlmonkey 2 is injected with PBS. Monkeys (1 and 3-5) vaccinated with GAB-1are tested positive for anti-GAB-1 antibody and appear healthy afterbeing challenged with WR virus. The control monkey 2 is tested negativefor anti-GAB-1 antibody and appears sick after being challenged with WRvirus.

In Table II, monkeys 1 and 3-5 showed increased antibody titers againstGAB-1 antibody, WR antibody, and vaccinia WR neutralizing antibody,compared to monkey No. 2.

In Table III, patients immunized with GAB-1 produces higher antibodytiters than patients immunized with MVA E2. This shows that the GAB-1virus is more immunogenic than MVA E2.

In FIG. 1A, CEF, BSC-1 and VERO cells were infected with GAB-1 or wildtype Western Reserve (WR) virus at 0.05 plaque-forming unit (pfu), andharvested at 24 and 48 hours post infection, to measure themultiplication of GAB-1 and WR viruses in CEF, BSC-1 and VERO cells.GAB-1 grows in CEF much more efficiently than in VERO or BSC-1.Surprisingly, GAB-1 grows more efferently than the wild type,unattenuated WR virus, in each cell type.

In FIG. 1B, viral yield was analyzed by titrating the different viruspreparations in BSC-1 and VERO cells. Plaques were stained with crystalviolet at 48 h post infection.

In FIG. 1C, the virulent wildtype WR virus can replicates in CEF and gooutside of the cells forming lytic plaques because it is not attenuated.In contrast, the GAB-1 virus is an attenuated virus which does not gooutside of the cell (does not form lytic plaques or holes) and can onlyform morphologic plaques.

In FIG. 2, DNA was extracted from 0, 20, 100 and 200 passages over theCEF cells. The GAB-1 virus lost many nucleotides. GAB-1 cannot grow inmost of human cells tested up to now, but can grow very efficiently inCEF cells.

In FIG. 3A, monkeys were injected with GAB-1 virus vaccine or PBSsolution in the upper arm. A pustule developed after 7 days at the siteof injection in animals vaccinated with GAB-1, but not in monkeyinjected with PBS. In FIG. 3B, one month after vaccination, monkeys werechallenged with wildtype WR virus. After one week, the control animalswere clearly very sick (PBS), while the vaccinated animal (GAB-1)remained healthy.

In FIG. 4, the aceto-white staining in the uterus reveals papillomavirusinfection in a patient, characterized as cervical intraepithelialneoplasia grade 2 (CIN 2). The patient is free of lesions at week 14after GAB-1 treatment.

In FIG. 5, CEF cells were infected with GAB-1 or MVA E2 recombinantvirus. After 24 and 48 hours viruses were collected and titrated. GAB-1virus produced more viral particles than did MVA E2 recombinant vaccine.Both GAB-1 and MVA E2 are attenuated and non-virulent.

Example 1

Construction of New Attenuated Vaccinia Virus GAB-1.

Cell and viruses. Monkey-kidney (VERO) and human carcinoma (HeLa) andBSC-1 cells were maintained in a humidified air-5% C0₂ atmosphere at 37°C. Chicken embryo fibroblast (CEF) cells were grown in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% fetal calf serum(Gibco BRL, Gaithesburg, Md.), 20 μM glutamine, and 50 units/mlpenicillin, and 50 μg/ml of streptomycin. The vaccinia virus strainsWestern Reserve (WR) and Lederle-Chorioallantoic were obtained from theAmerican Type Culture Collection (ATCC), Manassas, Va., USA). WR viruswas propagated in HeLa cells and the Lederle virus strain was grown inVERO cells. Immediately, BSC-1 cells were infected using the originalseed of vaccinia virus (Lederle-Chorioallantoic). After 48 hours ofinfection 10 different virus plaques were isolated. These virus plaqueswere grown, first in VERO cells and then in chicken embryo fibroblastcells (CEF). Two virus plaques were selected and about many passagesover CEF cells were performed. Two of these virus isolates were used toprepare two different stocks of the new GAB-1 virus.

Once the GAB-1 virus was prepared, we characterized virus replication ofGAB-1 in different permissive cell lines. BSC-1, VERO and CEF cells.These cells were infected with GAB-1 or WR viruses using 0.05 pfu.Virus-infected cells were collected at 24- and 48-hours post-infection.GAB-1 virus produced the double amount of virus as compared with the WRvirus in CEF cells (FIG. 1 A). We next analyzed the plaque morphology ofthe GAB-1 virus by infecting BSC-1 and VERO cells for 48 hours. GAB-1virus produced smaller plaques as compared with the WR virus (FIG. 1 B).In addition, CEF infected with GAB-1 produced morphologic plaques ascompared with the big lytic plaques formed by WR virus (FIG. 1C). Thisresult showed that the GAB-1 virus can replicate efficiently in CEFcells but not in VERO cells (FIG. 1 B and FIG. 1C).

Purification of Recombinant GAB-1 Viruses

Chicken fibroblast cells were grown to a confluence of 85% and theninfected with recombinant virus GAB-1 in DMEN medium containing 2.5%horse serum. Infection is induced using 1 virus for every 10 cells.Infection is left for one hour at 37° C. Infected cells are thenincubated for 48-72 hours at 37° C., in DMEM medium containing 5% horseserum and in an atmosphere of 4.5% CO₂. The infected cells are collectedand centrifuged at 2500 rpm for 10 minutes at room temperature.

These collected viruses are resuspended in 20 ml of DMEM-5% with horseserum. They are then frozen and thawed three times in order tocompletely break the cells and liberate the viruses. Rupture is verifiedwith the assistance of a microscope.

The solution is centrifuged at 2,500 rpm for 10 minutes and the virusesare resuspended in 10 ml of 10 mM Tri HCl, pH: 9 buffer. The viruses aresonicated for 2 minutes and placed in a Dounce homogenizer. The virusesare homogenized with 40 strokes and then centrifuged at 2,500 rpm for 10minutes. The supernatant containing the viruses is placed in a 37%sucrose bed. It is centrifuged in rotor SW28 at 13,500 rpm, 80 min, 4°C. The viruses located in the bottom of the tube is resuspended inphysiological saline solution of PBS, pH: 7.4. The viruses are stored inaliquots of 1 ml and kept at −70° C. The number of particles isinitially calculated measuring the absorbance of the virus solution at260 nm. A unit of virus at 260 nm is approximately 1.3×10¹⁰ virus/ml,which is approximately equal to 5×10⁸ live viruses. The number of viralparticles is also measured with the assistance of a specific antibodyagainst the vaccinia virus, using it in GAB-1-infected cells. In thisway live viruses can be counted as compared with infected cells.

Example 2

DNA Analysis of Consecutive Passages of Lederle Strain of Vaccinia VirusOver CEF Cells

Viruses from 0, 20, 100 and 200 passages of Lederle virus were grown inCEF cells using Dulbecco modified Eagle medium (DEMEM) (Gibco BRL,Gaithesburg, Md.) supplemented with 10% horse serum (Gibco BRL,Gaithesburg, Md.). Cells were maintained in a humidified air-5% CO₂atmosphere at 37° C. DNA virus from different passages were isolated byphenol-chloroform extraction followed by ethanol precipitation. DNA wasquantified by densitometry and analysed by agarose gel 0.4%. (FIG. 2).It was found that by passing the Lederle strain into CEF cells the viruswas losing several thousands of nucleotides from its genome. (FIG. 2).

Example 3

Safety of the GAB-1 Vaccinia Virus.

GAB-1 attenuated virus fully protects monkeys against a lethal challengewith the WR virus.

In order to test the safety, efficacy and potency of the GAB-1 virus,four monkeys' captive-bred adult (5-13 years) healthy male monkeys(Cercopithecus aethiops) were intradermally immunized with 10⁵ pfu ofthe GAB-1 virus and one monkey injected with physiological solution(PBS). All monkeys were kept and maintained in negatively pressurized P3facilities following good principles of laboratory animal care. Monkeyswere immunized by the multiple-puncture technique using a bifurcatedneedle as described (Frey et al., 2002). The injections were done byperforming 15 strokes directly into the skin of the upper arm. It wasobserved that in vaccinated animals a small pustule of 0.3 cm ofdiameter appeared three days after injection. This pustule became redafter three days and finally dried by day fourteen and disappear. (FIG.3A) The vaccination discomfort was very mild at most because monkeys didnot present rash, or pain at the place of injection. One month afterimmunization animals were challenged intramuscularly with 10⁸ pfu of WRvirus. Half of this virus dose was injected into the triceps muscle, andthe other half into the femora's muscle. Animals immunized with GAB-1showed no evidence sickness, and remained clinically healthy for twomonths after challenge (FIG. 3 B). These monkeys did not change theirfood intake during the whole study. In contrast, the non-vaccinatedcontrol monkey showed a sick appearance and red spots in the skin afterthe fourth day of challenge. This animal stopped eating after the fifthday of challenge with an increase of temperature fever. He alsopresented cramps, weight lost and severe red lesions in extremities, dueto virus spreading. In contrast all GAB-1 vaccinated animals remainedhealthy all the time without the presence adverse side effects. (TableI). In addition, all immunized animals with GAB-1 virus generatedantibodies against the virus, showing that GAB-1 virus is a goodimmunogen. (Table II).

Example 4

Immune response generated in monkeys after GAB-1 immunization. Vacciniavirus antibody responses against GAB-1 and WR were analyzed in animalsvaccinated with GAB-1. Briefly, ELISA plates were coated with either5×10⁵ purified virus particles of WR or GAB-1 virus. Sera from animalswere collected at the beginning of vaccination and 4 weeks later afterimmunization. Serum dilutions were added to plates and incubatedovernight at 4° C. Plates were then washed three times with phosphatebuffer saline (PBS), and incubated with a 1/2000 dilution of horseradishperoxidase-conjugated Protein A (Sigma Aldrich) for 1 hr. Followingthree more washes, the plates were incubated with the peroxidasesubstrate 0-phenylene diamine (Sigma Aldrich) at room temperature during30 min. Absorbance was read at 405 nm on an ELISA plate reader (Bio-TekInstruments). All monkeys vaccinated with GAB-1 generated antibodiesagainst the virus. In addition, these antibodies also react against theWR virus. In contrast, a monkey (number 2) injected with PBS does notgenerated antibodies against vaccinia virus. These results showed thatthe GBA-1 virus can efficiently stimulate the immune system (Table II).

Neutralizing Antibody Responses in Monkeys after Immunization with theGAB-1 Virus.

Because the WR virus has a very similar genome-sequence to the variolavirus (smallpox) and because the GAB-1 vaccination protected animalsagainst challenge with WR virus. We decided to analyze the capacity ofthe antibodies generated in immunized animals with GAB-1 virus toneutralize the WR virus in infection experiments. Serial dilutions ofsera from GAB-1 vaccinated monkeys were mixed with WR virus andimmediately added to BSC-1 cells to allow viral infection. Infectiousviruses were detected by counting the number of plaques formed. Serafrom the four vaccinated animals can neutralize the WR virus (Table II).In contrast, antibodies from the un-vaccinated animal did not neutralizethe WR virus. Sera neutralizing titers were approximately 1/1000 in allanimals. These results showed that antibodies against the GAB-1 virusefficiently bind the WR virus and can prevent viral infection. Takentogether, these results show GAB-1 virus is a safe and efficient virusto stimulate the immune system.

Example 5

Study Design and Subjects.

Clinical Protocol for Testing the Potential of the GAB-1 Virus in theTreatment of Papillomavirus Lesions.

A phase I, II and III clinical protocol was conducted in whichapproximately 200 patients (male and female) with HPV intraepitheliallesions were treated with GAB-1 virus. Patients were recruited from thefollowing medical institutions in Mexico: Hospital de Cuautitlan, inEstado de Mexico; Hospital de la mujer, in Michoacan state; SanatorioSan Francisco, Hospital General de Veracruz, Hospital 20 de Noviembre,ISSTE, Hospital Militar, and Hospital de Nutricion, Instituto Nacionalde Cancerologia, in Veracruz state; and in Venezuela: InversionesMilfred Medical. Patients were admitted to the protocol once therelevant eligibility criteria had been met. These criteria includedpresence of papillomavirus (HPV), age between 14 and 60 years, not to bepregnant and have CIN 1, CIN 2, CIN 3 or condyloma lesions, includingseveral clinical laboratory exams, a complete physical exam, and theclinical history of the patient. The protocol has been approved by theEthics and scientific committee from the hospitals. Before starting thetreatment with GAB-1, the physician revised all these data and confirmedthat the patient was eligible for the protocol. We used 10⁵ GAB-1 viralparticles for each dose of the viral vaccine. The GAB-1 virus wasinjected directly into the uterus in a radial clock-wise fashion at 3,6, 9, and 12 o'clock once a week during six weeks for women; or directlyinto the urethra once a week during five weeks, for men. In cases whenlesions were visible, for example in vulva and anus, GAB-1 was alsoapplied locally at the base of each lesion. (FIG. 4).

We also analyzed the difference in immunogenicity between our previousMVA E2 recombinant virus vaccine and the present GAB-1 virus vaccine. Wefound that by using 100-fold less of the GAB-1 virus vaccine (10⁵ viralparticles) compared with the 10⁷ viral particles of the MVA E2 that webeen using in the past 20 years. W were able to eliminate all thepapilloma lesions present in the patients. These results show that theGAB-1 virus can stimulate very efficiently the immune system, and iscapable of completely eliminate all papillomavirus lesions in patientsby using much less virus as we did for the MVA E2 virus vaccine.

In addition, we compared the humoral immune response generated againstthe GAB-1 or the MVA E2 recombinant vaccine (Table III). We observedthat patients vaccinated with GAB-1 virus produced more antibodiesanti-vaccine than the MVA E2 recombinant virus. Surprisingly, GAB-1virus is much more immunogenic than the MVA E2 virus that has been usedto produce many vaccines for several virus infectious diseases.

Comparative Virus Production Between GAB-1 Virus and MVA E2 RecombinantVirus.

Chicken embryo fibroblast (CEF) were obtained from 11-day fertile eggs.Briefly, chicken body was minced and cells were obtained by addition of0.25% trypsin. Cells were harvested by centrifugation and resuspended inDulbecco's modified Eagle's medium (DMEM) supplemented with 10% horseserum (Gibco, BRL), 20 uM glutamine, 50 unit/ml penicillin and 50 ug ofstreptomycin in a humidified air-5% CO2 atmosphere at 370 C. After cellswere collected and attached to microcarriers (Cytodex) and grown is a15-liter Bioreactor Celligen-Plus. Cells were infected either with GAB-1or MVA E2 recombinant virus. The viruses were collected at 24 and 48hours after infection. Immediately the infected-cells were freeze-thawedthree times. Viruses were purified by two successive sucrose (40-10%)zonal centrifugation steps. The purified viruses were titrated on CEFand stored at −70° C.

Surprisingly, there was a big difference in virus yield productionbetween GAB-1 and MVA E2 recombinant virus. (FIG. 5). Under the sameconditions we obtained almost 50-100 times more GAB-1 virus than the MVAE2 recombinant virus. This stronger viral yield is a great advantage forvirus vaccine production for commercial purposes.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

Definitions

Gene: Refers to a sequence of DNA that carries the information forsynthesis of a polypeptide or protein.

Infection: Refers to the penetration of an infectious agent into a cell,and if the conditions are sufficient, the microorganism can bereplicated within the cell.

Expression systems: Systems in which foreign proteins can be synthesizedwithin bacteria or cells.

Through cloning of genes into expression vectors, it is possible bymeans of transfection techniques to introduce genes into cells andbacteria and for these genes to be efficiently expressed. These vectorscan be plasmids or recombinant viruses normally introduced intobacteria, insect cells, yeast cells, or animal cells.

For example, if the protein is expressed in a bacterial system, a highquantity can be produced. However, there is a drawback: if the proteinis unstable, it may rapidly degrade, and the yield would be very low.Another disadvantage is that if eukaryotic genes are expressed in thebacterium, and the protein requires post-translational modifications(glycosylations for example), then a non-functional protein issynthesized. Expressing these genes in systems such as yeasts oreukaryotic cells solves the problem.

Yeast systems are very efficient, since these cells grow at roomtemperature and do not require expenditure of energy to raise theirtemperature. But as with bacteria, they do not have all thepost-translational modification systems, which means that they also havelimitations.

Viral expression systems such as baculovirus (a virus that infectsinsect cells) are used a great deal today, since they can express genesefficiently and have the advantage that they can excrete the proteininto the external environment, facilitating its purification. However,as there are only two or three glycosylation systems, not all proteinsexpressed in this system are functional.

Viral vectors such as poxvirus can be excellent expression vectors. Forexample, the vaccinia virus belonging to this family has been used forthe expression of many biologically interesting proteins, and since itis expressed in mammalian cells, the proteins maintain their normalbiological activity.

An even more innovative step is the use of the vaccinia virus to expressexogenous proteins that in turn serve as immunogenic agent for use asvaccinations against some diseases or infectious agent in particular.Also, the use of these vectors is safe, since they do not cause adverseeffects on people or animals that are vaccinated. This has already beendescribed and demonstrated with a great number of vaccines used to fightdiseases such as prostate cancer; hepatitis A, B, and C; rabies, HIV,mumps, flu, parainfluenza, malaria, tuberculosis, or against plasmodium,hantaan virus, SARS, and breast cancer. In all cases the vaccine'sefficacy has been validated through experiments with laboratory animals.

Samples of GAB-1 virus have been placed on deposit under the terms ofthe Budapest Agreement at Collecion de Microorganismos del CNRG(Boulevard de la Biodiversidad No. 400, Col Rancho las Cruces CP.47600,Tepatitlan de Morelos, Jalisco, Mexico) as GAB-1 VIRUS DE LA VACCINIAATENUADO and under Accession No. CM-CNRG TB167. The deposit was made onSep. 22, 2020.

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What is claimed is:
 1. A viral preparation, comprising an attenuatedLederle-Chorioallantoic strain of vaccinia virus that has been seriallypassaged in a host cell.
 2. The viral preparation of claim 1, whereinthe host cell is selected from the group consisting of chicken embryofibroblast (CEF) cells, a monkey kidney cell line (VERO), and a monkeyepithelial cell line (BSC-1).
 3. The viral preparation of claim 1,wherein the attenuated Lederle-Chorioallantoic strain of vaccinia virusproduces more viral particles in an animal cell than does a wildtypeWestern Reserve strain of vaccinia virus.
 4. The viral preparation ofclaim 1, wherein the attenuated Lederle-Chorioallantoic strain ofvaccinia virus produces morphologic plaques, but not lytic plaques inchicken embryo fibroblast (CEF).
 5. The viral preparation of claim 1,wherein the attenuated Lederle-Chorioallantoic strain of vaccinia virushas been passaged in chicken embryo fibroblast (CEF) cells for at least100 passages.
 6. The viral preparation of claim 5, wherein theattenuated Lederle-Chorioallantoic strain of vaccinia virus has beenserially passaged in CEF cells for at least 200 passages.
 7. The viralpreparation of claim 1, wherein the attenuated Lederle-Chorioallantoicstrain of vaccinia virus has lost at least 5000 base pairs ofnucleotides after serial passaging.
 8. The viral preparation of claim 7,wherein the attenuated Lederle-Chorioallantoic strain of vaccinia virushas been passaged in chicken embryo fibroblast (CEF) cells for at least100 passages.
 9. The viral preparation of claim 7, wherein theattenuated Lederle-Chorioallantoic strain of vaccinia virus has lostbetween 8,000 and 12,000 base pairs of nucleotides after serialpassaging.
 10. The viral preparation of claim 9, wherein the attenuatedLederle-Chorioallantoic strain of vaccinia virus has been seriallypassaged in chicken embryo fibroblast (CEF) cells for at least 200passages.
 11. The viral preparation of claim 1, wherein the attenuatedLederle-Chorioallantoic strain of vaccinia virus protects an animal fromwildtype Western Reserve strain of vaccinia virus.
 12. The viralpreparation of claim 1, wherein the attenuated Lederle-Chorioallantoicstrain of vaccinia virus is at effective in reducing size of cervicalintraepithelial neoplasia by at least 80%.
 13. The viral preparation ofclaim 1, wherein the attenuated Lederle-Chorioallantoic strain ofvaccinia virus generates more viral particles in a host than doesmodified vaccinia Ankara (MVA) E2 strain of vaccinia virus.
 14. Theviral preparation of claim 1, wherein the Lederle-Chorioallantoic strainof vaccinia virus produces a higher antibody titer than does a modifiedvaccinia Ankara (MVA) E2 strain of vaccinia virus in a patient.
 15. Theviral preparation of claim 1, wherein the Lederle-Chorioallantoic strainof vaccinia virus produces a higher antibody titer than does wildtypeWestern Reserve strain of vaccinia virus in an animal.